目的 建立一种灵敏、快速的高细胞色素P450(CYPs)酶活性的大鼠原代肝细胞模型,用于评价经CYPs酶代谢后产生肝毒性的药物。方法 分别采用苯巴比妥和β-萘黄酮两种CYPs酶广谱诱导剂构建大鼠原代肝细胞模型;应用“cocktail”探针底物法考察两种诱导剂对CYPs酶的影响;以基于CYPs酶代谢导致肝毒性的药物他克林(TAC)、双氯芬酸钠(DIC)和对乙酰氨基酚(PAR)为模型药物,评价所构建的高CYPs酶活性大鼠原代肝细胞模型与普通大鼠原代肝细胞间灵敏性的差异;应用所构建的高CYPs酶活性大鼠原代肝细胞模型评价维拉帕米(VER)的肝毒性。结果 与普通大鼠原代肝细胞相比,3种模型药物在高CYPs酶活性大鼠原代肝细胞中,在较低剂量时可使细胞上清液中乳酸脱氢酶(LDH)、细胞中活性氧(ROS)水平升高,线粒体膜电位(MMP)水平下降,或相同剂量下得到更严重的损伤结果,CYPs酶抑制剂1-aminobenzotriazole(ABT)和metyrapone(MET)能抑制这3种损伤的发生;100 μmol/L维拉帕米在普通大鼠原代肝细胞中并未引起LDH、ROS和MMP的改变,但在高CYPs活性大鼠原代肝细胞模型中,会引起细胞损伤,ABT和MET能抑制这种损伤。结论 经诱导建立的两种高CYPs酶活性大鼠原代肝细胞模型能更灵敏的评价基于CYPs酶代谢致毒药物的肝毒性;两种诱导剂诱导得到的高CYPs酶活性大鼠原代肝细胞模型对经不同CYPs酶亚型代谢的药物评价结果有各自优势。应用高CYPs酶活性大鼠原代肝细胞模型证实维拉帕米的致毒机制是经CYPs酶代谢产生肝毒性。

Objective To establish a rapid and sensitive primary rat hepatocytes model with high cytochrome P450 enzyme (CYPs) activity for evaluating the drug-induced liver injury based on CYPs metabolism. Methods The primary rat hepatocytes model was established with CYPs nonspecific inducer phenobarbital and β-naphthoflavone; The “cocktail” probe method was applied to inspect the effect of inducers on CYP450 enzyme. Tacrine (TAC), diclofenac sodium (DIC), and paracetamol (PAR) which induced liver injury based on the metabolism of CYPs were as the model drugs. The difference and sensibility between primary rat hepatocytes model with high CYPs activity and general primary rat hepatocytes model were evaluated; The liver injury of verapamil (VER) was evaluated with the primary rat hepatocytes model with high CYPs activity. Results After administration with three kinds of model drugs (100 μmol/L) to primary rat hepatocytes with high CYPs activity, the levels of lactate dehydrogenase (LDH) in hepatocytes supernatant and the reactive oxygen species (ROS) in hepatocytes were increased, and the mitochondrial membrane potential (MMP) was decreased. Nonspecific CYPs inhibitors, 1-aminobenzotriazole (ABT) and metyrapone (MET) could suppress the occurrence of this liver injury; VER (100 μmol/L) could not lead to LDH, ROS and MMP change in general primary rat hepatocytes model, however, it could cause cytotoxicity in primary rat hepatocytes model with high CYPs activity. The CYPs inhibitors ABT and MET suppressed this damage. Conclusion The primary rat hepatocytes model with high CYPs activity is more sensitive and suitable to evaluate the hepatotoxicity of drugs based on metabolism of CYPs. Two types of primary rat hepatocytes models with high CYPs activity have respective advantages in evaluation of different types of drug. VER was confirmed to induce liver injury based on metabolism of CYPs with the application of primary rat hepatocytes model with high CYPs activity.

国家自然科学基金(No.81373890);教育部高等学校博士学科点专项科研基金(No.20121210110011);教育部长江学者与创新团队发展计划(No.IRT-14R41)